Purpose.:
Rho kinase (ROCK) is associated with VEGF-driven angiogenesis, as well as with inflammation and fibrosis. Therefore, the effect of AMA0428, a novel ROCK inhibitor, was studied in these processes, which highly contribute to the pathogenesis of neovascular AMD.

Results.:
The AMA0428 dose-dependently reduced proliferation and VEGF-induced migration of HUVEC and HBMEC (P < 0.05). No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were enhanced (P < 0.05) by AMA0428 administration. There was no apoptosis induction. The AMA0428 significantly reduced CNV and vessel leakage 2 weeks after laser treatment, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 42% (P < 0.05) and collagen deposition on day 30 by 43% (P < 0. 05), whereas DC101 had no effect on inflammation nor on fibrosis.

Conclusions.:
The results suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to VEGF suppression). These results point to a potential therapeutic benefit of ROCK inhibition in neovascular AMD.

Age-related macular degeneration (AMD) was first recognized approximately 130 years ago and has been extensively investigated over the past decades in an attempt to unravel the underlying disease mechanisms. However, the pathogenesis remains incompletely understood. Age-related macular degeneration is the most frequent cause of irreversible vision loss in the developed world1,2 and is becoming an important socio-medical problem, due to the increased life expectancy in the western world. Two different subgroups of AMD are classically distinguished: an atrophic lesion or dry form and an exudative, wet form. Dry AMD is the most common, and less sight-threatening type of macular degeneration.3 The crucial difference between the two subgroups is the development of choroidal neovascularization (CNV) in wet AMD,4 which causes rapid progression to significant sight loss.5

In addition to neovascularization, wet AMD is characterized by inflammation and fibrosis,6 processes that generally contribute to wound healing. Anti-VEGF administration is currently the most commonly used treatment option for active CNV.7,8 However, there is only limited evidence that anti-VEGF therapy might reduce fibrosis and inflammation.9,10 A major issue related to anti-VEGF therapy is that VEGF is a key player in vascular and neuronal outgrowth and maintenance,11 and its inhibition can result in blood vessel regression and neurodegenerative processes.12,13 The use of anti-VEGF may therefore give rise to local side effects, but also potentially to important systemic side effects, including cardiovascular effects, cerebrovascular accidents, and myocardial infarction.14 Moreover, conventional therapy that focuses primarily on inhibiting angiogenesis may not be optimal because of the inflammatory and fibrotic component in AMD. Therefore, new therapeutic strategies that address the different pathological processes are required to provide additional options to better manage AMD.

As our understanding of the pathophysiologic mechanism of AMD improves, the opportunity to refine treatment approaches increases. An attractive novel therapeutic option in the treatment of wet AMD is the inhibition of Rho kinase. To date, there are two Rho kinase isoforms that have been described, ROCK1 (ROKβ) and ROCK2 (ROKα).15 They regulate numerous cellular processes, such as cell proliferation, survival, permeability,16 endothelial cell (EC) polarity, and directed cell migration.17 The Rho/ROCK pathway also mediates vascular smooth muscle cell and pericyte recruitment,18 which is essential for blood vessel maturation and stability. Indeed, pericyte recruitment lags behind endothelial sprouting, leading to a window of pericyte absence that allows for vascular plasticity resulting in survival, growth, and remodeling, dependent on the presence of different growth factors such as VEGF.19 This process is responsible for the “leaky nature” of the proliferating vessels and, together with a breakdown of the blood-retinal barrier (BRB), causes edema formation.20 Importantly, Ren et al.21 demonstrated that cell spreading requires transient downregulation of RhoA activity. Defects in cell spreading and directional motility, present in the pathology of AMD, may therefore be rescued by ROCK inhibitors.20 Moreover, a potential advantage of ROCK inhibition, compared with anti-VEGF therapy, would be its additional effect on inflammation and fibrosis.22–26 Although AMD is not a predominantly inflammation-driven disease, unlike uveitis and scleritis, inflammation has been found to play a critical role in the disease pathogenesis and progression.27–29 Local inflammation accompanied by complement activation, cell lysis, and immune responsiveness are important facets of AMD pathogenesis and progression to wet AMD.30,31 Both innate immunity and autoimmune components are known to be involved in AMD development.27 As such, age-related drusen, a by-product of these chronic inflammatory events, accumulate between the RPE and Bruch's membrane. This will lead to recruitment of tissue-destructive macrophages, microglia, and inflammatory cell-derived cytokines and chemokines.32 Moreover, several diseases, such as wet AMD and diabetic retinopathy, which are characterized by macular edema, retinal and vitreous hemorrhage, and fibrovascular scarring, have a final common pathophysiological denominator, namely the retinal response to injury, with chronic wound healing leading to fibrosis.

In this study, AMA0428, a potent and novel ROCK inhibitor derived from recent work of Amakem Therapeutics was used (Ref. 33 and Boland S, oral communication of unpublished results, 2013). We focused on the three major processes involved in AMD: inflammation, angiogenesis, and fibrosis. Inhibition of ROCK could be an alternative/additive therapy for anti-VEGF in the treatment of wet AMD, without the systemic side effects of anti-VEGF.

All cell lines were routinely maintained at 37°C in a humidified atmosphere of 5% CO2 and medium was replaced every 2 to 3 days. Cells between third and sixth passage were used in all experiments. All in vitro assays were repeated at least three times.

Apoptosis Assay

Human brain microvascular ECs and HBVPs were trypsinized and were seeded in coated (0.1% gelatin for ECs; poly-L-lysine for pericytes) 96-well plates (Costar; Corning, Inc., Corning, NY, USA) at an initial density of 10 × 103 cells per well in 100 μL complete medium. Plates were incubated at 37°C in a humidified atmosphere of 5% CO2. Twenty-four hours later, the medium was replaced with fresh medium, with or without AMA0428 (0.5, 1, 2.5, 5, 10, 25 μM; Amakem Therapeutics, Diepenbeek, Belgium) or the equivalent of vehicle/dimethyl sulfoxide (DMSO; Sigma-Aldrich Corp., St. Louis, MO, USA). Two days later, Caspase-GloR 3/7 Reagent (Promega, Leiden, The Netherlands) was added to the cell suspension (1:1 ratio) and incubated at room temperature for 1 hour. Afterward, the supernatant was transferred into a white-bottom 96-well plate (Thermo Scientific, Roskilde, Denmark), in which the luminescence of each sample was measured in a plate-reading luminometer (Microplate Luminometer LB96V, EG&G Berthold, VIC, Australia).

Proliferation Assay

Subconfluent HBMECs, HUVECs, and HBVPs were trypsinized and were seeded at 37°C in coated 96-well plates at an initial density of 3 × 103 cells per well in 100 μL complete medium. The following day, cells were incubated with AMA0428 or DMSO at different concentrations (0.5, 1, 2.5, 5 μM) in their respective complete media. For HBMECs and HUVECs, administration of an anti-VEGF antibody (bevacizumab [Avastin]; Genentech/Roche Therapeutic, Welwyn Garden City, UK), at a concentration of 2.5 mg/mL and 5.0 mg/mL, was used as a positive control in full medium. In serum-free medium, EC were challenged with recombinant human VEGF165 (rhVEGF165; 100 ng/mL or 5.22nM; R&D Systems, Minneapolis, MN, USA) to increase proliferation and treated with AMA0428 (0.5, 1, 2.5, 5 μM) or bevacizumab (2.5 mg/mL or 16.8 μM, 5 mg/mL or 33.6 μM) as positive control.

For HBVPs, recombinant human platelet-derived growth factor (rhPDGF-BB; R&D Systems) was used as a positive control and rhVEGF165 (R&D Systems) was added as a negative control. In another series of experiments, HBVPs were serum starved (medium supplemented with 0.1% FBS) overnight, 6 hours after cell seeding. The medium of the cells was changed to fresh serum-free medium containing AMA0428 or DMSO at different concentrations (0.5, 1, 2.5, 5 μM).

Forty-eight hours after growth factor or compound administration, cell proliferation was assessed in all experiments using the WST-1 Cell Proliferation Assay System (Roche, Mannheim, Germany). Complete or serum-free medium was used as control.

Scratch Assay

The effect on cell migration was evaluated using an in vitro scratch assay, performed as previously described.34 Human brain microvascular endothelial cells and HBVPs were grown to confluence in 48-well tissue culture dishes and were serum starved overnight. The next day, the monolayer was scratched with a plastic pipette tip to remove the confluent cells by two perpendicular linear scratches to create linear wounds. The HBMECs were incubated at 37°C in starvation medium with or without AMA0428 (0.5–5 μM), DMSO, and/or 20 ng/mL rhVEGF165. Recombinant human PDGF-BB (20 and 40 ng/mL) and/or AMA0428 (0.5–5 μM), was added to HBVP. After 2 hours, the movement of cells into the wound area was measured using a crossline micrometer on a Primo Vert 1 microscope (both from Zeiss, Oberkochen, Germany). The shortest distance between the edges of migrated cells (including protrusions) from both sides was measured to determine the effect of AMA0428 after wounding of a monolayer of cells. These measurements were at four predefined points at start and at 2, 4, 6, 8, and 24 hours after wounding.35

Under-Agarose Migration Assay

To investigate the effect of ROCK inhibition on pericyte recruitment, an under-agarose migration assay36 was performed as described. Briefly, 0.48 g ultrapure agarose was suspended in 10 mL double-distilled water (ddH2O). The agarose solution was briefly heated in a microwave until it boiled. Next, 2% of the agarose solution was added to 1% FBS EGM-2. This solution was added into tissue culture dishes, and was allowed to solidify at room temperature. Afterward, two 5-mm wells were punched in the gels at 1 mm distance from each other. The gels were equilibrated at 37°C, 5% CO2 for 1 hour. Next, any condensation that might have formed in the wells was removed and twenty thousand HUVEC were plated in one well in 20 μL EGM-2 medium with 1% FBS. After 3 hours the HUVEC were treated with AMA0428 in different concentrations (1, 2.5, 5 μM) or the equivalent of DMSO. Thirty minutes later, HBVPs (2 × 104 cells in the same medium) were added to the other 5-mm well. The distance of HBVP migration toward the HUVEC was measured with a crossline micrometer in a Primo Vert 1 microscope (both from Zeiss) every day for 4 successive days.

Permeability Assay

Endothelial cell permeability was determined by measuring the passage of FITC-labeled dextran through a tight monolayer of cells. For this, HUVECs were seeded at a concentration of 24,000 cells/transwell on 0.2% gelatin-coated transwell membranes (Corning 0.4-μm polyester membrane, CLS3470; Sigma-Aldrich Corp.). After 2 days, a tight monolayer of cells was formed and the transwell inserts were transferred to a new 24-well plate. The culture medium was supplemented with vehicle, VEGF165 (2.08 μM = 40 ng/mL, V7259; Sigma-Aldrich Corp.) with/without AMA0428 (0.01, 0.1, 1 μM). Next, 70 kDa FITC-dextran (1 mg/mL, FD70S; Sigma-Aldrich Corp.) was added on top of the cells and medium samples were collected from the lower compartment every 15 minutes for 1 hour. Fluorescence intensity was evaluated using a Fluostar Omega V1.30 microplate reader (BMG Labtech, Ortenberg, Germany). Data of three independent experiments (±SEM) are presented as mean relative values compared to VEGF treatment.

Animals

Male C57BL/6J mice (8–10 weeks old) were obtained from Charles River Laboratories, L'Arbresle Cedex, France, and were used in all experiments. Housing and all experimental animal procedures were approved by the Institutional Animal Care and Research Advisory Committee of the KU Leuven. All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the 2010/63/EU Directive for animal experiments, using the guidelines of our own institution.

Compounds

In this study, the ROCK inhibitor AMA0428 was tested in comparison to vehicle (both provided by Amakem NV). For vehicle, H2O-PEG was used (PEG 400; Fagron NV, Waregem, Belgium). First, a safety assessment was made by intravitreally injecting 1 μL of the highest dose of the compound (1000 ng) and compared with noninjected/naïve mice, sham (puncture, no fluid injection) and vehicle-injected mice (10 mice/group; only one eye per mouse was used). The potential side effects were examined at day 14 post injection on hematoxylin and eosin (H&E)–stained retinal sections by measuring the total retinal thickness, the thickness of the outer and inner nuclear/plexiform layer, and photoreceptor layer. Also, retinal ganglion cell density was quantified two times on the same sections by counting ganglion cell nuclei at 500 μm on four sections on each side of the optic nerve head.

Murine Model of CNV

Mice were anesthetized by an intraperitoneal (IP) injection of 60 mg/kg sodium pentobarbital (Nembutal, CEVA; Sante Animale, Brussels, Belgium) and their pupils dilated with tropicamide (Tropicol; Thea, Clermont-Ferrand, France). As described in literature,37–39 three laser burns were placed with a 532-nm green laser (Ophthalas 532 EyeLite Laser Phothocoagulator; Alcon Surgical, Puurs, Belgium) at the 9, 12, and 3 o'clock positions around the optic disk using a slit lamp delivery system with a hand-held cover slide as a contact lens and Genteal Gel (Novartis, Vilvoorde, Belgium). Each spot had a size of 50 μm, laser duration of 100 ms, and a power of 400 mW, based on previous experiments in our laboratory.7 Bubble production as a sign for the rupture of the Bruch's membrane was necessary to include the spot. In all experiments, one eye per mouse was lasered, except for the expression studies (ELISA), where two eyes per mouse received laser treatment to obtain sufficient tissue.

Expression of ROCK, VEGF, and Placental Growth Factor (PlGF).

A slight variation of the CNV model, using 30 separate laser spots on Bruch's membrane, was performed to investigate the expression of ROCK, VEGF, and PlGF in the injured choroid and retina. Mice were divided into one naïve group (nonlasered, nontreated) and three lasered groups (five mice per group; two eyes were lasered): a first nontreated group, a second AMA0428 (1000 ng) group, and a third vehicle group. Mice were treated at day 0 and day 2 via intravitreal (IVT) injections. Three days after laser treatment, the eyes were enucleated and the retina–choroid–sclera complexes were microdissected and snap-frozen. The levels of ROCK, VEGF, and PlGF were determined by ELISA. To obtain sufficient protein concentrations, each sample contained pooled tissue of two posterior segment complexes of one mouse. Cell lysates from these segments were obtained using the Mammalian Cell Lysis kit (Sigma-Aldrich Corp.) and expression levels were determined with the Quantikine ELISA kit for mouse VEGF and PlGF-2 (both from R&D Systems) and the Rho-associated kinase (ROCK) Activity ELISA Assay (Merck Millipore, Overijse, Belgium), with detection limit of 7.8 pg/mL, 1.49 pg/mL, and 100 nU, respectively.

Treatment Regimen.

Immediately after laser treatment, C57BL/6J mice were divided into different groups and their eye was consequently injected IVT by using an analytic science syringe (SGE Analytic Science, Crownhill, Milton Keynes, UK) and glass capillaries with a diameter of 50 to 70 μm at the end, controlled by the UMP3I Microsyringe Injector and Micro-4 Controller (all from World Precision Instruments, Inc., Hertfordshire, UK). Before injection, the eye was anesthetized by topical instillation of 0.4% oxybuprocaine (Unicaine; Théa Pharma, Schaffhausen, Switzerland). One group was treated with the ROCK inhibitor, AMA0428 (1 μL; 100–1000 ng); the second group was used as control and received an injection of vehicle (H2O-PEG; 1 μL). In the positive control groups, the eye was treated IVT respectively with the well-described rat anti-mouse VEGF-R2 antibody, DC10140,41 (1 μL, 6200 ng) or an isotype-matched control antibody (1C8, 1 μL, 4800 ng; both provided by ThromboGenics NV, Heverlee, Belgium).

In the first experiment, repeated IVT injections of the compound/control antibodies were given at similar concentrations on days 0, 4, 10, and 20 after laser treatment. These time points were based on previously performed IVT injections by Van de Veire et al.7 Mice were killed on day 5 (to evaluate inflammation), day 14 (to evaluate angiogenesis), or day 30 (to evaluate fibrosis) after laser treatment. In a second experiment, a single IVT injection with AMA0428 or vehicle was given on day 0 or day 3 and mice were killed on either day 5 or day 14 after laser treatment. In a last experiment, a combination of the most effective dose of AMA0428 and a suboptimal dose of DC101 (anti–VEGF-R2) was tested. For this, a dose-response curve was first established for both compounds. Based on these results, a dose of 1550 ng DC101 (25% of full concentration) was chosen and a dose of 1000 ng AMA0428 (full dose). All the experimental groups consisted of 10 mice per group (unless stated otherwise).

(Immuno)histochemistry

Mice were anesthetized with an IP injection of Nembutal and killed by cervical dislocation. Afterward, both eyes were enucleated and fixed in 1% paraformaldehyde (PFA) overnight.

To analyze the effect of AMA0428 on inflammatory cell infiltration in the CNV lesions, the retina was removed from the dissected posterior segments. These posterior eye cups, which included RPE, the choroid, and the sclera, were stored in PBS. To stain all leukocytes, a rat anti-mouse CD45 antibody (1:100; Pharmingen, Erembodegem, Belgium) was used overnight, diluted in Tris-buffered saline (TBS)-Triton 0.3%. The following day, the tissues were incubated for 2 hours with rabbit anti-rat biotin (RaRat-B)-labeled antibody (1:300; DakoCytomation A/S, Copenhagen, Denmark), diluted in TBS-Triton 0.3%. Antibody binding was visualized by fluorescent staining using streptavidin-Alexa-568 (1:200; Molecular Probes, Life Technologies, Eugene, OR, USA) in TBS-Triton 0.3% for 2 hours. The flat mounts of the posterior eye cups were mounted with Prolong Gold with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes).

To investigate the effect of AMA0428 on angiogenesis, retrobulbar perfusion with 200 μL FITC-conjugated dextran (50 mg/mL, Mr 2 × 106 Da; Sigma-Aldrich Corp.),42 was performed and after 2 minutes mice were killed. Vessel leakage was assessed, as previously described,7 by retrobulbar injection using a total volume of 200 μL per mouse, of which 100 μL Texas Red-conjugated Dextran 70 kDa (5 mg/mL; Molecular Probes) together with 100 μL FITC-labeled lectin (1 mg/mL, Lycopersicon esculentum; Vector Laboratories, Inc., Burlingame, CA, USA). Five minutes later, the red dye was washed out by intracardial perfusion with NaCl 0.9% for 5 minutes, to remove the tracer form the actual blood vessels. All eyes were enucleated and fixed overnight in 1% PFA. The next day, all RPE–choroid–sclera complexes were dissected and flatmounted on a slide containing a drop of Vectashield mounting medium (Vector Laboratories) to prevent the fluorescent dyes from bleaching.

Deposition of collagen was analyzed by Sirius Red staining and collagen I immunostaining on paraffin-embedded eyes. Serial sections were cut at 7-μm thickness in five series on five glass slides. First, H&E staining was performed to localize the laser spots. For the Sirius Red and collagen I staining, the consecutive slides were used. Slides were deparaffinized, washed, and placed in Sirius Red solution (Direct Red 80 and 1.3% picric acid solution; both Sigma-Aldrich Corp.) for 60 minutes. Sections were then placed in 0.01N HCl (Prolabo, Amsterdam, The Netherlands) for 2 minutes, dehydrated and mounted with DPX mounting medium (Prosan, Gent, Belgium). Collagen I staining was also performed on deparaffinized sections. After antigen retrieval (Dako Antigen Retrieval Solution; Dako, Heverlee, Belgium), endogenous peroxidase activity (0.3% hydrogen peroxide in methanol for 20 minutes) and aspecific binding was blocked using pre-immune goat serum (DakoCytomation A/S). Next, sections were incubated with rabbit anti-collagen I (1:500 in tris/NaCl blocking reagent; Abcam, Cambridge, UK) overnight. As secondary antibody, RaRat-B (DakoCytomation A/S) was used. The bound antibodies were visualized using signal amplification with the Perkin Elmer kit (Renaissance TSA Indirect, Waltham, MA, USA) with cyanine 3 as fluorophore. Afterward, the slides were mounted with Prolong Gold containing DAPI (Molecular Probes).

Microscopic Analysis

Using a microscope (Leica Microsystems, Wetzlar, Germany) equipped with a digital camera (Axiocam MrC5; Zeiss), images were analyzed at a magnification of ×20 and a resolution of 2584 × 1936 pixels. Commercial software (KS300; Zeiss) was used for morphometric analyses, as previously described.43–45 The area taken by immunopositive leukocytes and blood vessels was quantified by calculating respectively the CD45-positive and the FITC-dextran-positive CNV area in the samples. Deposition of collagen was determined by first defining the middle of each spot on H&E staining and secondly by measuring the Sirius Red-positive or collagen I-positive area on five serial sections in the middle of each laser spot. Sirius Red was analyzed under polarized light to distinguish mature from immature collagen fibers. For each staining, the average area of typically three laser spots per eye was used of 10 mice (n = 10), generating 10 data points (only one eye per animal was used) per group, based on literature.46 However, lesions were excluded (1) if there were signs of eye infection during the experiment, excluding the entire eye; (2) if there was hemorrhage in the eye, excluding all three lesions of one eye; (3) if the lesion had fused with another lesion, excluding those two lesions; or (4) if the lesion was an outlier, meaning that the CNV area exceeded 100,000 μm2 and was more than five times larger than the next largest lesion in the eye; or when the CNV area was “too small,” that is, it was less than one-fifth the area of the next smallest lesion in the eye. These exclusion criteria were based on the study performed by Poor et al.46

To analyze for leaky vessels, confocal imaging was performed, with a Laser Scanning Microscope (LSM; Olympus, Melville, NY, USA). Laser scanning microscope–scanning software FluoView (FV10-ASW; Olympus) was used for digital acquisition and processing of the images. Vessel leakage was quantified by calculating the leaky area (Texas Red-conjugated Dextran; red area) as a proportion to the blood vessel area (FITC-lectin; green area). An average of 11 frames per final image/spot were used.

Statistical Analysis

All statistical data were generated in GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA). A one-way ANOVA with vehicle as comparator was performed on all in vivo immunomorphometric data with a Dunnet's post hoc analysis test, as previously reported.46 For the expression studies, a one-way ANOVA was also performed with the lasered, nontreated group as comparator. In vitro cell apoptosis and proliferation was analyzed by a one-way ANOVA. The migration assays were evaluated using mixed model analysis for repeated measures (two-way ANOVA). Data are represented as mean ± SEM, unless otherwise stated, and considered to be statistically significant at P ≤ 0.05. All experiments were analyzed blindly and randomized.

Results

AMA0428 Inhibits Proliferation of ECs, but Not of Pericytes

To investigate the direct effect of AMA0428 on cell proliferation, HUVECs, HBMECs, and HBVPs were cultured. First, possible toxic effects of the ROCK inhibitor were checked using a caspase 3/7 apoptosis assay. Hereto, the cells were incubated in complete medium with AMA0428 or vehicle/DMSO at different concentrations. There was no significant increase in apoptosis after 48 hours when treated with AMA0428 from 0.5 μM up to 25 μM for EC and up to 10 μM for HBVPs, as compared with the cells treated with the equivalent of DMSO or control medium (data not shown). Thus, AMA0428 did not induce caspase 3/7 activity, suggesting that there are no obvious toxic effects at the used doses.

To determine the effect of AMA0428 on proliferation of HBMECs and HUVECs, cells were grown in complete medium or in serum-free medium to which rhVEGF165 was added. In both conditions, AMA0428 (0.5, 1, 2.5, and 5 μM) induced a significant decrease in cell proliferation of HBMECs, as compared with vehicle/DMSO solutions in, respectively, complete/full medium or serum-free medium enriched with rhVEGF165 (P < 0.05 for all concentrations; Figs. 1A, 1B). Also, the growth of HUVECs was inhibited in both conditions after AMA0428 (0.5, 1, 2.5, and 5 μM) administration, as compared with the respective control solutions (P < 0.05 for all concentrations; Figs. 1C, 1D). The VEGF inhibitor, bevacizumab (Avastin; Roche), which was used as a positive control in a concentration of 2.5 mg/mL and 5 mg/mL,43,47 reduced EC growth comparable to AMA0428 (Figs. 1A–D).

Next, the effect of the ROCK inhibitor, AMA0428, on pericyte proliferation was investigated using HBVPs. Analysis of the WST-1 proliferation assay showed no significant difference in number of HBVPs after treatment with AMA0428 (Figs. 1E, 1F) as compared with DMSO-treated cells (data not shown). As previously described,48 administration of rhVEGF165 resulted in a significantly decreased HBVP cell growth. On the contrary, rhPDGF significantly increased pericyte proliferation in starvation medium (Figs. 1E, 1F).

The effect of AMA0428 on cell migration was evaluated using an in vitro scratch assay.34 All cells were incubated in serum-free medium overnight. Immediately following scratch injury, HBMEC and HBVP were treated with AMA0428 vehicle (DMSO) and/or with rhVEGF165 (20 ng/mL) or PDGF (20 ng/mL). Sole administration of AMA0428 did not inhibit nor stimulate migration of HBMECs. On the other hand, VEGF administration led to near complete (90.0% ± 3.5%) wound closure in HBMECs at 8 hours after scratching. Furthermore, addition of AMA0428 to VEGF-stimulated HBMECs significantly blocked migration compared with vehicle (overall P < 0.05; Fig. 2A). In HBVPs, VEGF administration inhibited migration, whereas PDGF or AMA0428 treatment led to near complete wound closure after 8 hours, respectively to 85.0% ± 4.2% and to 85.0% ± 4.5%, as compared with DMSO (overall P < 0.05; Fig. 2C). The level of wound closure in the DMSO solution did not significantly differ from serum-free medium after 8 hours in both assays. These data were confirmed using the under-agarose migration assay.36 Analysis showed that, in comparison to vehicle (DMSO), treatment with AMA0428 significantly stimulated migration of HBVPs toward HUVECs (overall P < 0.05 for all concentrations; Fig. 3). To determine whether this effect was not solely induced by exposure of HBVPs to AMA0428, as HBVP migration was increased after AMA0428 administration in the scratch assay, the under-agarose migration assay was performed with and without HUVECs. These experiments revealed an additional stimulatory effect on migration by AMA0428 in the presence of HUVECs (data not shown).

Thus, administration of AMA0428 was able to inhibit migration of VEGF-stimulated ECs and to stimulate pericyte recruitment. Overall, these data indicate that AMA0428 has the potential to reduce new blood vessel formation, on the one hand by inhibiting EC proliferation and migration and, on the other hand, by stimulating pericyte recruitment, thereby promoting maturation of blood vessels.

AMA0428 Decreases EC Permeability In Vitro

Endothelial cell permeability was determined by measuring the passage of FITC-labeled dextran through a tight monolayer of HUVECs. Cells were treated with vehicle or AMA0428 with or without VEGF165 (2.08 μM = 40 ng/mL). Cell permeability was determined by evaluating fluorescence intensity every 15 minutes for 1 hour. A significant decrease in permeability of EC monolayers was observed after treatment with AMA0428 at 15 and 30 minutes after VEGF administration (0.1 μM; P < 0.05; Fig. 4).

Upregulation of ROCK, VEGF, and PlGF in the CNV Mouse Model Is Inhibited by AMA0428

To investigate whether ROCK signaling contributes to CNV in vivo, we first analyzed levels of ROCK, VEGF, and PlGF in choroidal tissue of mice that received 30 laser spots. This procedure is known to induce the upregulation of growth factors such as VEGF49 and PlGF.7,50 Enzyme-linked immunosorbent assay for ROCK, VEGF, and PlGF showed a 2-fold increase in ROCK activity, a 1.5-fold increase in VEGF levels, and 2.3-fold increase in PlGF levels after laser treatment compared with naïve nonlasered mice, suggesting that these molecules are involved in the pathogenesis of CNV. The upregulation of these factors could be blocked significantly by the IVT administration of AMA0428 (P < 0.05; Fig. 5). Of note, the results of the vehicle group were comparable to the lasered, nontreated group (data not shown).

The possible in vivo efficacy of IVT administered AMA0428 was determined in the laser-induced murine model for CNV. In a first approach, the potential side effect of AMA0428 (1000 ng) on retinal degeneration was investigated. Hereto, repeated injections did not reveal any gross changes in the thickness of the neuronal cell layers and in number of retinal ganglion cells in the AMA0428-treated eyes on H&E staining (Fig. 6). These data suggest no significant observable toxicity of AMA0428 to the neurosensory retina.

The AMA0428 (1000 ng) seems safe by IVT injection in naïve mice. (A) Compared to vehicle, sham injection, or naïve mice, IVT administration of AMA0428 did not alter the total retinal thickness, the thickness of the outer and inner nuclear and plexiform layer, or photoreceptor layer as investigated on H&E staining. (B) The AMA0428 did not reduce the retinal ganglion cell number compared to vehicle, sham, or naïve mice. Data are represented as mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points.

Figure 6

The AMA0428 (1000 ng) seems safe by IVT injection in naïve mice. (A) Compared to vehicle, sham injection, or naïve mice, IVT administration of AMA0428 did not alter the total retinal thickness, the thickness of the outer and inner nuclear and plexiform layer, or photoreceptor layer as investigated on H&E staining. (B) The AMA0428 did not reduce the retinal ganglion cell number compared to vehicle, sham, or naïve mice. Data are represented as mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points.

Next, to compare the effect of this new ROCK inhibitor with a reference treatment, additional groups of mice were injected IVT with the murine anti–VEGF-R2 antibody, DC101, or with an irrelevant mouse antibody (1C8). To unravel which disease processes AMA0428 could affect, mice were killed at defined time points: at the peak of inflammation (day 5), angiogenesis (day 14), and fibrosis (day 30).7

Inflammation is known to be involved in the earliest changes of CNV.6 A CD45 immunostaining was performed to study the effect of ROCK inhibition on inflammation. We compared the effect of repeated IVT injections of AMA0428 (1000 ng) versus DC101 on leukocyte infiltration in the laser-injured eyes. After 5 days, repeated administration of AMA0428 reduced the CD45-positive area in the lesions by 31.0% ± 11.8%, as compared to vehicle-treated eyes (n = 10 eyes; data not shown), whereas DC101 did not affect the inflammatory process. To investigate whether single administration would be sufficient, one IVT injection was given on day 0 or on day 3. Either AMA0428 1000 ng or 100 ng reduced inflammation, respectively, by 42.0% ± 13.1% and 38.0% ± 6.7% compared with vehicle-treated eyes (n = 10 eyes; P < 0.05; Fig. 7A) when supplied at the day of injury. Inflammation was also decreased after a single injection on day 3 with AMA0428 1000 ng or 100 ng, respectively, by 44.0% ± 14.4% and 38.0% ± 16.6% (n = 10 eyes; P < 0.05; Fig. 7B). These results showed that a single IVT injection on day 0 with AMA0428 was the most effective. Of note: A single injection on day 0 or day 3 with DC101 did not result in a significant reduction of leukocytes (n = 10 eyes; P > 0.05; Figs. 7A, 7B).

Intravitreal administration of AMA0428 reduces inflammation in the CNV mouse model. (A) A significant inhibition of inflammation was seen with a single IVT injection on day 0 with AMA0428 (100 ng: P < 0.01; 1000 ng: P < 0.001). (B) A single IVT injection on day 3 with AMA0428 gave a significant reduction of leukocytes at day 5 (100 ng: P < 0.05; 1000 ng: P < 0.01). Early and late administration of DC101 had no effect on inflammation in the CNV model (P > 0.05). Data represent mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points. (C) Representative pictures of CD45 whole-mount staining (injection day 0) of eyes treated with vehicle or AMA0428 (1000 ng). Scale bars: 50 μm.

Figure 7

Intravitreal administration of AMA0428 reduces inflammation in the CNV mouse model. (A) A significant inhibition of inflammation was seen with a single IVT injection on day 0 with AMA0428 (100 ng: P < 0.01; 1000 ng: P < 0.001). (B) A single IVT injection on day 3 with AMA0428 gave a significant reduction of leukocytes at day 5 (100 ng: P < 0.05; 1000 ng: P < 0.01). Early and late administration of DC101 had no effect on inflammation in the CNV model (P > 0.05). Data represent mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points. (C) Representative pictures of CD45 whole-mount staining (injection day 0) of eyes treated with vehicle or AMA0428 (1000 ng). Scale bars: 50 μm.

To confirm the antiangiogenic capacities of AMA0428 in vivo, neovascularization was investigated at 14 days after laser treatment via FITC-dextran perfusion. At a dose of 1000 ng and 100 ng, repeated administration of AMA0428 reduced the total vessel area, respectively, by 79.0% ± 21.2% and 57.0% ± 21.0% (n = 10 eyes, P < 0.001, Figs. 8A, 8B), 14 days after laser treatment. Similarly, repeated injection of DC101 inhibited vessel area by 75.0% ± 17.3% (n = 10 eyes; P < 0.001), with no significant differences between the two compounds (P > 0.05, Fig. 8A). A single IVT injection on day 0, immediately after laser treatment, with AMA0428 1000 ng or 100 ng was less effective, because it only inhibited angiogenesis by, respectively, 55.0% ± 22.6% and by 34.0% ± 9.9% (n = 10 eyes; P < 0.05; data not shown). A single injection on day 3 gave a reduction of the active angiogenic process of 39.0% ± 15.9% for AMA0428 1000 ng (n = 10 eyes; P < 0.05), which was less potent compared with the injection on day 0 (data not shown).

The effect of AMA0428 on the integrity of the BRB was also investigated at day 14 using a vessel leakage assay with Texas Red-conjugated Dextran and FITC-labeled lectin perfusion. The AMA0428 (1000 ng) blocked choroidal vessel leakage by 29.0% ± 13.9% (n = 10 eyes, P < 0.05; Figs. 8C, 8D) when administered IVT on day 0, day 4, and day 10. The effect was similar to that induced by repeated injections of DC101, namely 22.0% ± 12.3% (n = 10 eyes, P < 0.05; Fig. 8C).

Choroidal neovascularization is also characterized by fibrosis. Therefore, Sirius Red staining and collagen I staining were used to check the effect of Rho kinase inhibition on the process of collagen deposition in vivo. Repeated (days 0, 4, 10, 20) IVT injections of AMA0428 1000 ng and 100 ng reduced collagen deposition in the lesions at day 30 by 34.0% ± 10.2% and 25.0% ± 5.9%, respectively (n = 10 eyes; P < 0.05, Sirius Red staining; data not shown), whereas DC101 did not reduce fibrosis 30 days after laser treatment (n = 10 eyes; P > 0.05). In addition, a collagen I staining was used to confirm the fibrotic reaction, as this type of collagen is most widely expressed in the CNV model.51 Analysis gave similar results compared with the Sirius Red staining, namely a reduction of 43.0% ± 9.9% and 39.0% ± 9.9% when treated, respectively, with 1000 ng and 100 ng AMA0428 (n = 10 eyes; P < 0.05; Figs. 9A, 9B).

The AMA0428 inhibits fibrosis 30 days after laser treatment in the CNV mouse model. (A) Administration of repeated (days 0, 4, 10, 20) IVT injections with AMA0428 decreased fibrosis significantly compared to vehicle (P < 0.05), whereas DC101 did not affect fibrosis. Data are represented as mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points. (B) Representative pictures of Collagen I staining of eyes treated with vehicle or AMA0428 (1000 ng). Borders of the CNV lesion are marked by a dashed line, the immunopositive area inside the dashed line represents the new fibrotic area, which was used for analysis. Scale bars: 50 μm.

Figure 9

The AMA0428 inhibits fibrosis 30 days after laser treatment in the CNV mouse model. (A) Administration of repeated (days 0, 4, 10, 20) IVT injections with AMA0428 decreased fibrosis significantly compared to vehicle (P < 0.05), whereas DC101 did not affect fibrosis. Data are represented as mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points. (B) Representative pictures of Collagen I staining of eyes treated with vehicle or AMA0428 (1000 ng). Borders of the CNV lesion are marked by a dashed line, the immunopositive area inside the dashed line represents the new fibrotic area, which was used for analysis. Scale bars: 50 μm.

Because complications of anti-VEGF treatment are mostly dose-dependent,13,52–54 the possibility to partially substitute DC101 by AMA0428 was explored. Therefore, first a dose-response experiment was set up for both AMA0428 and DC101 (Figs. 10A, 10B). Based on these results, a combination of the most effective dose of AMA0428 and a suboptimal dose of anti–VEGF-R2 (25%) was given. This low dose of anti–VEGF-R2 was chosen to leave a larger window for the substitution effect. The combination therapy was given IVT on day 0, day 4, and day 10, and mice were killed on day 14. Neovascularization was reduced by 83.0% ± 11.4% with the combination therapy (n = 10 eyes; P < 0.001), that is, comparable to the highest dose of anti–VEGF-R2 therapy alone (75.0% ± 17.3% reduction; n = 10 eyes; P < 0.001; Fig. 10C).

Thus, by adding AMA0428, the dose of DC101 can be reduced by 4-fold, while maintaining the efficacy of the full dose of anti-VEGF therapy. This opens the possibility to reduce the adverse effects sporadically linked to vascular and neuronal regression caused by VEGF inhibition.12,13

Discussion

The effect of a novel ROCK inhibitor (AMA0428) on inflammation, angiogenesis, vascular leakage, and fibrosis, processes known to be involved in neovascular AMD, was investigated. Our results demonstrate that AMA0428 was effective in reducing these different processes in vitro and in the in vivo laser-induced CNV mouse model. Moreover, we showed that this ROCK inhibitor had a more profound effect compared with VEGF-R2 inhibition (DC101). Our previous findings have consistently demonstrated that DC101 is as effective as bevacizumab, the reference treatment in clinical practice, in mouse models of CNV, oxygen-induced retinopathy, and glaucoma filtration surgery, thus confirming its suitability for translational ophthalmological research.55

The present study indeed indicates that administration of AMA0428 might reduce the formation of new blood vessels by inhibiting EC proliferation and VEGF-mediated migration. These results are comparable to published data of Y-27632, another reference ROCK inhibitor.56,57 Because it is known that VEGF-induced changes in the endothelial F-actin cytoskeleton require activation of Rho kinase,58 disruption of RhoA/ROCK signaling might inhibit VEGF-mediated alterations to cytoskeletal morphology.18,59,60 Aepfelbacher et al.61 described some evidence for a role of ROCK in EC migration, based on experiments performed in the presence of an endothelial growth supplement.

Moreover, AMA0428 might also improve vessel maturation, by stimulating pericyte recruitment, as shown by the in vitro under-agarose migration assay. Previous studies showed that exposure of human vascular pericytes to different ROCK inhibitors led to a migratory phenotype in various assays.18,56 A previous study also demonstrated that activated polymorphonuclear neutrophils induced pericyte relaxation in the cremaster muscles of mice by inhibiting the RhoA-ROCK pathway.18 Overall, it is known that ROCK inhibitors decrease myosin light chain phosphorylation in pericytes and induce loss of stress fibers and focal adhesions,18,62,63 thereby facilitating migration and thus recruitment of pericytes.

To obtain our results in vivo, we used the standard laser-induced mouse model for CNV, as this model is widely used to investigate wet AMD.37–39 As anti-VEGF therapy is an important treatment modality in this pathology, we directly compared the in vivo effect of AMA0428 to the well-established VEGF-R2 antibody, DC101. We demonstrated for the first time that ROCK inhibition is as effective as DC101 in reducing neovascularization, with an additional inhibitory effect on inflammation and fibrosis in the CNV mouse model. Besides a clear decrease in ROCK activity, administration of AMA0428 also resulted in a reduced VEGF and PlGF upregulation in CNV lesions. Although a direct effect of ROCK inhibition on VEGF or PlGF levels cannot be excluded, it seems unlikely, as VEGF and PlGF are upstream of ROCK; however, the interconnection between the pathways is still unclear and needs to be unraveled further. Therefore, we rather hypothesize that the reduced expression of both growth factors can be explained by an indirect effect of the working mechanism of the ROCK inhibitor. Indeed, the immediate action of AMA0428 on neovascularization and inflammation can lead to a diminished production of additional growth factors.

It is described that anti-VEGF therapy reduces the inflammatory response in the in vivo CNV model, through a VEGF-R1 effect.64 However, when both VEGF-R1 and 2 are blocked, an accumulation of inflammatory cells is seen at the CNV surface.50 In the clinical setting, anti-VEGF ligands affecting all receptors may thus induce inflammation in patients with neovascular AMD, in part due to an upregulation of different proinflammatory growth factors, such as PlGF.7,65 It is indeed known that PlGF is increased in the CNV mouse model,7,50 and after treatment with bevacizumab and different receptor tyrosin kinase inhibitors in colorectal, renal, and glioblastoma patients.66–68 In the present study, we showed that anti–VEGF-R2 therapy did not affect the inflammatory process in the CNV model, whereas AMA0428 was able to reduce inflammation after laser. The PlGF pathway is known to interact with the ROCK pathway, which had modulating effects on cell motility.69,70 Therefore, the anti-inflammatory effect of AMA0428 can partially be explained by the inhibition of PlGF upregulation in the CNV model. The Rho/ROCK pathway is also a key mediator in several other inflammatory processes involved in retinopathies, such as TNF-α signaling,71 anchoring of leukocytes72,73 and transcription of inflammatory cytokines (e.g., IL-6, IL-8) through nuclear factor–κβ activation.74,75 Therefore, ROCK inhibition might be a new way to address the underlying inflammatory pathophysiology of several retinopathies.

Fluid accumulation in the posterior retina, known as macular edema, is also involved in wet AMD and is the major cause of severe visual loss in neovascular AMD patients. This could result from a breakdown of the existing BRB at the level of the Bruch's membrane or from the “leaky nature” of the proliferating choroidal vessels. Anti-VEGF treatment can exert an effect on macular exudation, but might have only a limited and short-acting impact. Importantly, we showed that AMA0428 reduced vessel leakage. Moreover, IOP elevation has been described after IVT anti-VEGF administration, resulting in neuronal retinal damage.76 An additional likely benefit is that ROCK inhibitors, such as AMA0076, Y-27632, Y-39983, and others are known to have IOP-lowering capacities.77,78 We indeed confirmed IOP reduction with AMA0428 in mice and rabbits after topical installation (data not shown).

Although several approaches have been extensively investigated to inhibit promoters of angiogenesis in AMD,79 efforts to minimize fibrosis have met with limited success. Local destruction of neuronal cells in the retina, due to fibrosis, leads to reduction in function and, thus, in vision. We showed that IVT administration of AMA0428 was effective in reducing fibrosis, whereas anti–VEGF-R2 had no clear effect on the fibrotic response in the CNV model. From the literature it is known that the Rho/ROCK pathway is a downstream signaling mechanism of major ocular degenerative and fibrotic disease drivers, such as TGF-β,80,81 suggesting that it represents a new possible therapeutic target for fibrotic-related diseases. Inhibiting the initial inflammatory process and the subsequent angiogenesis might also indirectly decrease fibrosis, the final stage of CNV. Cao et al.82 already demonstrated that VEGF Trap, an inhibitor of VEGF and PlGF, was able to prevent fibrosis associated with CNV,82 due to its inhibition of leukocyte infiltration and angiogenesis. Acting on the inflammatory process to block collagen deposition is of utmost importance, as proinflammatory factors contribute to the development of experimental CNV and are present in human CNV fibrotic membranes.65

In a subpopulation of patients,83 the currently used VEGF inhibitors are known to cause adverse effects linked to vascular and neuronal regression in many organs, including the eye.13,52–54,83 These complications are related to the dose of anti-VEGF treatment and, thus, lowering this dose would be beneficial for patient outcome. Therefore, we explored whether we could substitute part of the anti–VEGF-R2 dose with AMA0428, while maintaining the efficacy of the full-dose anti–VEGF-R2. Importantly, our results indicated that the combination therapy of AMA0428 and anti–VEGF-R2 was effective in reducing neovascularization; however, the overall inhibitory effect of AMA0428 and the combination therapy was similar. Therefore, the combination of a lower dose of AMA0428 that does not give maximal inhibition, and various doses of DC101 can be considered in future studies to determine an additive effect between two compounds. Nevertheless, our data clearly reveal that ROCK inhibition might be a new approach as alternative or additive to anti-VEGF in the treatment of neovascular AMD patients.

The improvement of effective therapy for AMD will become even more significant as the population ages and as the prevalence of AMD and vision loss becomes more and more widespread. Inhibition of ROCK might improve current treatment options and thus help to fight the expected exponential rise in number of legally blind patients. The AMA0428 is an inhibitor of Rho kinases, which distinguishes itself from other agents by its efficacy profile and pleiotropic working mechanism.

Conclusions

In summary, targeting ROCK with AMA0428, a novel potent Rho kinase inhibitor, efficiently treated CNV in the laser-induced mouse model. Compared with anti-VEGF therapy, AMA0428 not only reduced neoangiogenesis but also blocked inflammation and fibrosis in the CNV lesions. These findings make ROCK inhibitors promising new candidates with potential additional therapeutic benefit in the treatment of neovascular AMD.

Acknowledgments

The authors thank Sofie Beckers, Ann Verbeek, and Martine Leijssen for their technical support. The AMA0428 was kindly provided by Amakem Therapeutics; DC101 and 1C8 were kindly provided by ThromboGenics NV.

The AMA0428 (1000 ng) seems safe by IVT injection in naïve mice. (A) Compared to vehicle, sham injection, or naïve mice, IVT administration of AMA0428 did not alter the total retinal thickness, the thickness of the outer and inner nuclear and plexiform layer, or photoreceptor layer as investigated on H&E staining. (B) The AMA0428 did not reduce the retinal ganglion cell number compared to vehicle, sham, or naïve mice. Data are represented as mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points.

Figure 6

The AMA0428 (1000 ng) seems safe by IVT injection in naïve mice. (A) Compared to vehicle, sham injection, or naïve mice, IVT administration of AMA0428 did not alter the total retinal thickness, the thickness of the outer and inner nuclear and plexiform layer, or photoreceptor layer as investigated on H&E staining. (B) The AMA0428 did not reduce the retinal ganglion cell number compared to vehicle, sham, or naïve mice. Data are represented as mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points.

Intravitreal administration of AMA0428 reduces inflammation in the CNV mouse model. (A) A significant inhibition of inflammation was seen with a single IVT injection on day 0 with AMA0428 (100 ng: P < 0.01; 1000 ng: P < 0.001). (B) A single IVT injection on day 3 with AMA0428 gave a significant reduction of leukocytes at day 5 (100 ng: P < 0.05; 1000 ng: P < 0.01). Early and late administration of DC101 had no effect on inflammation in the CNV model (P > 0.05). Data represent mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points. (C) Representative pictures of CD45 whole-mount staining (injection day 0) of eyes treated with vehicle or AMA0428 (1000 ng). Scale bars: 50 μm.

Figure 7

Intravitreal administration of AMA0428 reduces inflammation in the CNV mouse model. (A) A significant inhibition of inflammation was seen with a single IVT injection on day 0 with AMA0428 (100 ng: P < 0.01; 1000 ng: P < 0.001). (B) A single IVT injection on day 3 with AMA0428 gave a significant reduction of leukocytes at day 5 (100 ng: P < 0.05; 1000 ng: P < 0.01). Early and late administration of DC101 had no effect on inflammation in the CNV model (P > 0.05). Data represent mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points. (C) Representative pictures of CD45 whole-mount staining (injection day 0) of eyes treated with vehicle or AMA0428 (1000 ng). Scale bars: 50 μm.

The AMA0428 inhibits fibrosis 30 days after laser treatment in the CNV mouse model. (A) Administration of repeated (days 0, 4, 10, 20) IVT injections with AMA0428 decreased fibrosis significantly compared to vehicle (P < 0.05), whereas DC101 did not affect fibrosis. Data are represented as mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points. (B) Representative pictures of Collagen I staining of eyes treated with vehicle or AMA0428 (1000 ng). Borders of the CNV lesion are marked by a dashed line, the immunopositive area inside the dashed line represents the new fibrotic area, which was used for analysis. Scale bars: 50 μm.

Figure 9

The AMA0428 inhibits fibrosis 30 days after laser treatment in the CNV mouse model. (A) Administration of repeated (days 0, 4, 10, 20) IVT injections with AMA0428 decreased fibrosis significantly compared to vehicle (P < 0.05), whereas DC101 did not affect fibrosis. Data are represented as mean ± SEM. Numbers in the bars represent the used CNV lesions, which were averaged for 10 data points. (B) Representative pictures of Collagen I staining of eyes treated with vehicle or AMA0428 (1000 ng). Borders of the CNV lesion are marked by a dashed line, the immunopositive area inside the dashed line represents the new fibrotic area, which was used for analysis. Scale bars: 50 μm.